Natural killer cells are granular cytotoxic lymphocytes, derived from CD34+ hematopoietic progenitor cells (HPCs). They represent an essential component of the innate immune system. They comprise about 5 to 20% of lymphocytes in the spleen, liver, and peripheral blood and are also present—even if at lower frequencies—in the bone marrow, the thymus, and in lymph nodes. They were originally identified by their ability to kill certain (tumor-) target cells without sensitization. This killing works in vivo and in vitro and is not restricted by the target cell's expression of major histocompatibility complex (MHC) molecules. NK cells also possess natural cytotoxic activity against conspicious, such as but not restricted to (virus-) infected and/or tumor, cells. In addition, they mediate antibody-dependent cellular cytotoxicity (ADCC) of targets through FccRIII (CD16), a receptor that binds the Fc portion of antibodies.
In general, the traditional identifier for human NK cells is the absence of the T cell receptor complex (TCR, CD3), along with the expression of CD56, a 140-kDa isoform of neural cell adhesion molecule (NCAM). Based on their CD56 receptor expression density, human NK cells are often further subdivided into CD56dim or CD56bright NK cells. In the periphery, the majority (>90%) of NK cells have been found to consist of CD56dim along with high expression of CD16, and the remaining 10% are CD56bright NK cells coming along with low or no expression of CD16.
The described CD56dim NK cell fraction is generally considered the “classical cytotoxic NK cell subset”. The CD56bright fraction displays much lower cytotoxicity and, instead, produces high amounts of cytokines, including IFNγ and TNFα, indicating a primary role in immunoregulatory function.
The measurement of the cellular components in the blood is generally considered easier than that of other organs, since the cells are (at least in the periphery) not adherent or matrixed in a scaffolded organ. However, this is only partially true, since with the current methods, which mostly use the surface expression of so called CD (cluster of differentiation) antigens, it still remains challenging to determine the cell types in clinical routine applications. This is because for the cell sorting analysis as commonly used the cell samples need to be freshly isolated or immediately fixated in order to keep the cell entities intact. The blood/immunological methods used for blood component measurement for blood cells present in other tissues, including solid tissues at or after inflammation, and or the growth of solid tumors are limited, since they represent at most semi-quantitative methods (of particular relevance is the immunohistochemistry). The identification of specific epigenetic markers will greatly facilitate the clinical routine application of the measurement of blood cell types.
Even though almost all cells in an individual contain the exact same complement of DNA code, higher organisms must impose and maintain different patterns of gene expression in the various tissue types. Most gene regulation is transitory, depending on the current state of the cell and changes in external stimuli. Persistent regulation, on the other hand, is a primary role of epigenetics—heritable regulatory patterns that do not alter the basic genetic coding of the DNA. DNA methylation is the archetypical form of epigenetic regulation; it serves as the stable memory for cells and performs a crucial role in maintaining the long-term identity of various cell types.
The primary target of methylation is the two-nucleotide sequence Cytosine-Guanine (a ‘CpG site’); within this context cytosine (C) can undergo a simple chemical modification to become 5-methyl-cytosine. In the human genome, the CG sequence is much rarer than expected except in certain relatively dense clusters called ‘CpG islands’. CpG islands are frequently associated with gene promoters, and it has been estimated that more than half of the human genes have CpG islands (Antequera and Bird, Proc Natl Acad Sci USA. 90:11995-9, 1993).
Aberrant methylation of DNA frequently accompanies the transformation from healthy to cancerous cells. Among the observed effects are genome-wide hypomethylation, increased methylation of tumour suppressor genes and hypomethylation of many oncogenes (reviewed by Jones and Laird, Nature Genetics 21:163-167, 1999; Esteller, Oncogene 21:5427-5440, 2002; Laird, Nature Reviews/Cancer 3:253-266, 2003). Methylation profiles have been recognised to be tumour specific (i.e., changes in the methylation pattern of particular genes or even individual CpGs are diagnostic of particular tumour types) and there is now an extensive collection of diagnostic markers for bladder, breast, colon, oesophagus, stomach, liver, lung, and prostate cancers (summarized by Laird, Nature Reviews/Cancer 3:253-266, 2003).
EP 1213360 describes a method of identifying a cell, tissue or nucleus, comprising collecting information on the methylation pattern of DNA isolated from the cell, tissue or nucleus and analyzing the resultant information.
WO 2004/050706 describes a sub-group of T-cells, and relates to characteristics of regulatory T-cells which define them as such. The application also describes the uses of such T-cells, compositions comprising them and chemokines which recruit them in the modulation of an immune response.
Finally, EP 1826279 describes a method, in particular an in vitro method for identifying FoxP3-positive regulatory T cells, preferably CD25+ CD4+ regulatory T cells of a mammal, comprising analyzing the methylation status of at least one CpG position in the gene foxp3 or an orthologous or paralogous gene thereof, and the use of DNA-methylation analysis of the gene of the transcription factor FoxP3 for a detection and quality assurance and control of regulatory T cells.
In view of the above, it is an object of the present invention, to provide an improved method based on DNA methylation analysis as a superior tool in order to more conveniently and reliably identify NK cells and all different subsets of that cell type. Measurement can be done independent of purification, storage and to quite some extend also to tissue quality.
In a first aspect, the invention solves the above problem by providing a method for identifying natural killer cells in a sample derived from a mammal, comprising analysing the methylation status of at least one CpG position in one or more of the regions of one or more genes selected from NKG7, CX3CR1, FGR and GNLY, wherein a demethylation of at least one CpG position to at least 70% in said sample is indicative for a CD56 expressing natural killer cell. In a preferred embodiment, said natural killer cells of said mammal are preferably CD3−, non T-lymphocyte derived NK cells, but in certain embodiments also encompass CD3+ NKT cells.
In particular, methods of the invention are preferred, wherein said at least one CpG position in said sample is demethylated to more than 80% and preferably more than 90% and most preferred more than 95%.
A further embodiment of the invention then comprises the inventive method, wherein said at least one CpG position is present in the 5′ region upstream from the transcription start, promoter region, the 5′ or 3′ untranslated regions, intron, and/or exon/intron border or in the 3′ region downstream of the transcriptional stop. The invention provides that said at least one CpG position is preferably selected from the CpG positions of any of the genes CX3CR1 according to SEQ ID NO: 1, preferably selected from the CpG positions of the amplicon CX3CR1-1 (1452) according to SEQ ID NO: 5 or CX3CR1 amplicons ROI956 to 966, according to SEQ ID NOs: 6 to 16; FGR according to SEQ ID NO: 2, preferably of the amplicons FGR-1 (Amp. 1454) according to SEQ ID NO: 17 or FGR amplicons ROI967-977 according to SEQ ID NOs: 18 to 28; GNLY according to SEQ ID NO: 3, preferably of the amplicons GNLY 1 (1458) according to SEQ ID NO: 29 or GNLY amplicons ROI978 to 982 according to SEQ ID NOs: 30 to 34 and/or NKG7 according to SEQ ID NO: 4, preferably of the amplicons NKG7-1 (1455) according to SEQ ID NO: 35 or NKG7 amplicons ROI983 to 988 according to SEQ ID NOs: 36 to 41.
Yet another aspect relates to a method according to the present invention, wherein the analysis of the methylation status comprises a method selected from methylation specific enzymatic digests, bisulphite sequencing, analysis selected from promoter methylation, CpG island methylation, MSP, HeavyMethyl, MethyLight, Ms-SNuPE or other methods relying on a detection of amplified DNA. Also preferred is an additional analysis of the marker CD56, CD16 and/or CD8.
In particular, the inventors regard the herein described methods to be suitable for routine application, for example on a DNA-chip. Samples are selected from a fresh, fresh-frozen or fully prepared (such as formalin fixed paraffin embedded) sample, including mammalian body fluid, preferable human blood samples, serum samples or tumourous or non-tumourous solid tissue samples, organ or cell type blood sample, a sample of blood lymphocytes or a fraction thereof. These samples should be mammalian, preferably mouse, rat, monkey or human. Especially preferred is a mammal, most preferred a human, which suffers from or is likely to suffer from autoimmune diseases, viral or bacterial infections, transplant rejections, cancer, and/or allergy or any disease directly correlated to NK cells, such as—including but not limited to—diseases as phenotypically described by SCID-X1.
Another embodiment of the invention relates to the above methods, wherein said identification comprises a distinction and, optionally, a further quantification, of said natural killer cells from all major peripheral blood cell types or non-blood cells, and then further comprises the step of concluding on the immune status of said mammal based on said natural killer cells as identified. Hereby, in a sample of a mammal, including whole blood or various subfractions as well as tissues or isolated subfractions of tissues, NK cells can be identified and quantified due to their (unique) methylation pattern in the analysed genes. Based on this they can be quantitated.
Another aspect then relates to a method of the invention, wherein a demethylation of at least one CpG position in a first gene selected from NKG7, CX3CR1, FGR and GNLY in combination with a demethylation of at least one CpG position of at least a second gene selected from NKG7, CX3CR1, FGR and GNLY is indicative for a CD56dim or CD56bright natural killer cell. A preferred aspect then relates to a method of the invention, wherein a demethylation of at least one CpG position of NKG7 to at least 70% in combination with a demethylation of at least one CpG position of a gene selected from CX3CR1, FGR and GNLY to at least 70% in said sample is indicative for a CD56dim or CD56bright or CD8+ or CD8− natural killer cell.
In a further aspect the inventive method is useful for monitoring the level of CD56 expressing natural killer cells, in particular CD56dim or CD56bright, and/or CD16+ or CD16−, and/or CD8+ or CD8− natural killer cells in a mammal, comprising a method according to the invention, and comparing the amount of natural killer cells as identified to an earlier sample taken from the same mammal, and/or to a control sample.
In another aspect of the present invention, the method is also useful for measuring and/or monitoring the amount of said natural killer cells in response to chemical and/or biological substances that are provided to said mammal.
In yet another aspect, the invention provides an amplicon according to SEQ ID NOs: 5 to 41 or an amplicon produced by a primer-pair according to SEQ ID NOs: 42 to 181, and/or an oligomer hybridizing to a sequence selected from SEQ ID NOs: 1 to 41, preferably an oligomer selected from SEQ ID NOs: 42 to 181.
The invention also provides a kit for identifying and/or monitoring natural killer cells, in particular CD56dim or CD56bright, and/or CD16+ or CD16−, and/or CD8+ or CD8− natural killer cells, in a mammal based on the analysis of the methylation status of CpG positions in one or more genes selected from CX3CR1, FGR, NKG7 and GNLY, comprising materials for performing a method according to the invention.
Such an inventive kit comprises, but is not limited to, a) a bisulfite reagent, and b) materials for the methylation analysis of CpG positions selected from the CpG positions of the gene CX3CR1 according to SEQ ID NO: 1, preferably selected from the CpG positions of the amplicon CX3CR1-1 (1452) according to SEQ ID NO: 5 or CX3CR1 amplicons ROI956-966, according to SEQ ID NOs: 6-16; FGR according to SEQ ID NO: 2, preferably of the amplicons FGR-1 (Amp. 1454) according to SEQ ID NO: 17 or FGR amplicons ROI967-977 according to SEQ ID NOs: 18-28; GNLY according to SEQ ID NO: 3, preferably of the amplicons GNLY 1 (1458) according to SEQ ID NO: 29 or GNLY amplicons ROI978-982 according to SEQ ID NOs: 30-34 and/or NKG7 according to SEQ ID NO: 4, preferably of the amplicons NKG7-1 (1455) according to SEQ ID NO: 35 or NKG7 amplicons ROI983-988 according to SEQ ID NOs: 36-41.